The use of microfluidic structures in cytometry has rapidly increased with the advent of many microfabrication technologies capable of producing networks of fluidic circuits. Many cytometry applications in microfluidics attempt to differentiate cell types or cells with specific features within a population of cells. The detectors available to classify cells range from light scatter detection, to fluorescence marker, chromatic markers, and morphological image and feature differentiation. Detection necessarily precedes sorting and is followed by a coordinated directing of a cell into one pathway or another. Conventionally, high speed cell sorting fluid droplets that contain the cells of interest are ejected and electrically charged so that they may be electrostatically deflected using a high voltage field to direct the droplets to be collected in one of two locations for further processing.
Sorting cells has many purposes including further study of concentrated cell populations with similar features, such as stem cells or cells with a particular genetic or chemical characteristic that can be marked with fluorescence or stain. Sorting is also an effective means of validating a detection scheme in which a human observation or other reference instrument can evaluate cells with a given detection scheme mechanism or marker.
One sorting technique is found in U.S. Pat. No. 6,778,724, issued Aug. 17, 2004, to Wang et al. entitled, “Optical Switching and Sorting of Biological Samples and Microparticles Transported in a Microfluidic Device, Including Integrated Biochip Devices.” There disclosed is a method for switching and sorting small particles pushed with optical pressure forces, with laser light, as arises from VCSELs operating in Laguerre-Gaussian mode, at branching junctions in microfluidic channels so as to enter into selected downstream branches, thereby realizing particle switching and sorting, including in parallel.
Another sorting technique is found in U.S. Pat. No. 6,540,895 issued Apr. 1, 2003, to Spence, et al. entitled, “Microfabricated Cell Sorter for Chemical and Biological Materials.” There disclosed is a method for sorting cells into an appropriate branch channel based on the presence or amount of a detectable signal such as an optical signal, with or without stimulation, such as exposure to light in order to promote fluorescence. A thin cantilever may be included within a branch point, such that it may be displaced towards one or the other wall of the main channel, typically by electrostatic attraction, thus closing off a selected branch channel.
Sorting particles, such as cells, in a microfluidic channel takes advantage of the ability to use small fluid sample sizes of less than 1 μL and allows the detection and subsequent separation of sample particles into one of a plurality of possible pathways. The sorting mechanism remains contained and very close to the detection site, thus eliminating the need for long fluidic paths that dilute samples requiring extra processing steps to further concentrate the sample for subsequent detection or analysis.
Another advantage of a microfluidic approach is that sample carryover can be completely eliminated from hardware by providing low cost replacement fluidic pathways for each sample processed. The complexity and uncertainty of cleaning fluidics between sample processing is an often overlooked system detail requiring often 5 to 20 times the fluid flushed through tubing to clean it as it takes to process a sample. Cleaning is further complicated when using microchannels that force laminar flow conditions that eliminate the possibility of creating turbulent shear forces strong enough to clean tubing walls. The only mechanism of removing contamination from tubing walls is diffusion of the wall contaminant into a rinsing solution. Replacing rather than cleaning fluid paths requires less fluid and substantially improves certainty of eliminating sample cross contamination. The primary source of failure in fluidic instrumentation is found in the basic fluidics. Such fluidic failure modes include leaks, clogging, failed seals, biofilm growth, or accumulated contamination. Cleaning solution makes up the overwhelming biowaste volume from instruments such as flow cytometers.
However, until the present invention, no one has contemplated using a laminar flow channel in a microfluidic sorting system including a particle detection system, sorting control and coaxial cantilever injector. The use of the coaxial cantilever injector allows, for the first time, an ability to direct particles, including biological cells, into a selected one of a plurality of strata present in a laminar flow channel. The resultant sorted particles thus comprise an enriched sample for facilitating analysis of disease conditions including various cancers such as lung, colon, prostate, breast, cervical and ovarian cancers.